Characterization
The morphology and architectural characteristics of the NSs were
investigated using environmental scanning electron microscopy (ESEM)
(Quanta FEG 250 ESEM, FEI, USA). To avoid the coating on the sample
surface and thus retain the surface’s integrity, this ESEM was operated
in low vacuum mode. ESEM was used to thoroughly examine the inner and
exterior surfaces and cross section of all NS samples at different
magnifications. The chemical functionalities of the nutshell samples
were examined by a Bruker Tensor 27 FTIR spectrometer (PIKE
Technologies, USA) equipped with an attenuated total reflectance (ATR).
With multiple consecutive scans, the infrared spectra were gained in the
wavenumber range of 4000 - 400 cm⁻¹ at room temperature. A Confocal
laser scanning microscopy - CLSM (Zeiss LSM 510 confocal microscope)
imaging was carried out to examine the distribution of lignin,
cellulose, and hemicellulose within the cross-section of the WS. Cross
sections of the WS samples were sanded to a thickness of less than 50 µm
before the analyses. Lignin can be directly determined in the microscope
since it has the property of autofluorescence.[77]To reveal the cellulose and hemicellulose, the samples were treated with
Calcofluor White Stain (Sigma-Aldrich, Canada) and a 1 M KOH aqueous
solution (Sigma-Aldrich, Canada) for a duration of 5 minutes. Afterward,
the samples were stimulated with a laser emitting light at a wavelength
of 488 nm (detection range of 470 – 630 nm) to visualize lignin and at
a wavelength of 405 nm (detection range of 425 – 450 nm) to detect
cellulose and hemicellulose. Zeta potentials of the nutshells were
measured by using a Dyamic Light Scattering (DLS) - (Malvern Zetasizer
Nano ZS, UK). Prior to the zeta potential analysis, the nutshells were
ball-milled at 550 rpm for 30 minutes using a (Retsch planetary ball
mill PM 200 with agate and stainless-steel grinding jars) ball mill with
4 stainless steel (SS) balls. A 5% v/v suspension of each sample was
prepared in Milli-Q water. The zeta potential of each nutshell sample
was then determined three times for better reliability. The Optical
Contact Angle (OCA 35) instrument measured the water contact angle of
the surfaces. The surface area of each of the NSs was analyzed by
multipoint BET methods (Gemini VII 2390A, Micromeritics, USA). Samples
were degassed at 80 ºC for 10 h in nitrogen environment prior to the SA
analyses. Thermal Gravimetric Analyses (Q500, TA instruments, USA) were
conducted for the walnut shell samples in a nitrogen environment within
the temperature range from RT to 700 °C.